SUMMARY Due to the intensifying threat of the Zika virus to human health, the development of point-of-care methods to screen for the Zika virus represent an area of urgent need. Current approaches for the clinical testing of the Zika virus rely on conventional biochemical, genetic, and immunological laboratory assays that require complex instrumentation and expensive reagents (e.g., antibodies and fluorescent labels). While these methods are effective, the requirements of these methods, which also often involve multiple steps, making detection cumbersome, are prohibitive for point-of-care testing. The overall aim of this proposal is to develop a label-free screening platform for the detection of Zika by integrating stimuli-responsive and optically diffracting materials. Specifically, in this approach, we will investigate an optical biosensing platform comprised of a hydrogel material that swells in the presence a protease that is specific to Zika and is impregnated with a crystalline colloidal array (CCA). To develop such materials, polystyrene particles that self-assemble into a CCA will be co-polymerized into a polyacrylamide film that contains peptide crosslinks that may be cleaved by the protease (NS2B-NS3). Cleavage of the crosslinks by the protease is expected to physically alter the crosslinking density of the hydrogel network, thereby eliciting an increase in the volume of the hydrogel. As a result of swelling, the lattice spacing of the CCA will be altered, thereby triggering a change in optical diffraction of the hydrogel that can be detected at visible wavelengths without exogenous labels. The central hypothesis of this work is that the change in lattice spacing of the CCA-containing hydrogel, and thus the shift in wavelength of peak diffraction, will correlate with the proteolytic activity of NS2B-NS3. To directly test this hypothesis, the specific aims of this proposal are to: 1) characterize the optical response of CCA-containing hydrogels with peptide crosslinks in the presence of NS2B- NS3 (Aim 1) and 2) investigate the selectivity of the CCA-based sensing platform to NS2B-NS3 from the Zika virus (Aim 2). The sensitivity, including detection range and response time, of the films will be determined by treating the films, which will be prepared in multi-well plates that allow for high-throughput parallel detection, with different amounts of NS2B-NS3 and for different times. Additionally, a theoretical model of the swelling and optical responses will be developed, which will permit rational modification of the hydrogel properties to improve the overall sensitivity to NS2B-NS3. In aim 2, the selectivity of the screening platform towards the Zika homologue of NS2B-NS3 will be characterized by quantitatively comparing the optical response elicited by NS2B-NS3 from Zika with that from genetically related viruses. Furthermore, we will explore avenues to increase the selectivity of the sensing approach for Zika NS2B-NS3 by altering the peptide crosslinking sequence. In addition to point-of-care testing, the advantages of this label-free platform present new opportunities for screening for novel inhibitors of NS2B-NS3, which have immense therapeutic potential.